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Exercise 2: Diffusion, Osmosis and Membrane Permeability PreLab
In today's experiment, I predict, on the basis of Fick's law, that the order of the diffusion rates for the substances methylene blue, Alcian blue, ruthenium red, and methyl red (from the fastest to the slowest) will be
On the basis of Fick's law, I predict that the order of the diffusion rates for solutions of 0.3 mM methylene blue, 1 mM methylene blue, and 3 mM methylene blue (from the fastest to the slowest) will be
I predict that when red blood cells are placed in 0.9% NaCl, they will
I predict that when red blood cells are placed in deionized water, they will
I predict that when red blood cells are placed in 10% NaCl they will
What causes diffusion? What determines how rapidly it will proceed?
Saturday, January 24, 2009
How might diffusion rates affect the maximum size cells can attain. blood pressure is dangerously low. Choose the correct answer: At a given temperature. Why don't you choose sterile deionized water? Saturday.09% NaCl 0. isotonic. 2009 Page 18 7:05:05 PM . why is the maximum distance between a cell and the nearest capillary typically no greater than 0. Why can we use hemolysis rate as an indication of membrane permeability? 9. isotonic.09% NaCl 0. or. Indicate whether each of the solutions below is hypertonic. or hypotonic to mammalian red blood cells: 0. Choose the correct answer: A frog living in a freshwater pond is in an environment that is (hypertonic. You decide to administer an intravenous infusion to restore blood volume and blood pressure.9% NaCl 9% NaCl 12. hypotonic) to the frog's tissues. isosmotic. The patient had lost a good deal of blood. OSMOSIS. 14. & MEMBRANE PERMEABILITY — PRE-LAB 7. Assume that you are a health care practitioner who is treating an accident victim. or hyposmotic to each of the solutions below. and the patient is going into shock.01 mm? 13. Why is a hyperosmotic solution not necessarily hypertonic? 10. Indicate whether the intracellular fluid of mammalian red blood cells is hyperosmotic. 0. 8. Explain at the molecular level. the diffusion rate of large molecules is (greater than / less than) the diffusion rate of small molecules.9% NaCl 9% NaCl 11.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. January 24.
there are equal concentrations everywhere) they are at equilibrium and show no net tendency to move Saturday. Osmosis and Membrane Permeability PART I: DIFFUSION AND OSMOSIS INTRODUCTION Because of their kinetic energy. When molecules are distributed uniformly throughout a solution (that is.Exercise 2: Diffusion. January 24. the tendency of molecules to move from an area of high concentration to an area of low concentration. The pathways of molecules vibrating with kinetic energy are random (Fig. 2-1). This random motion is responsible for diffusion. The random path a molecule may take due to its inherent thermal energy. 2009 Page 19 7:05:05 PM . molecules are in constant motion unless they are at absolute zero (the temperature at which molecular motion ceases). Figure 2-1.
this would take a very long time. There are two important characteristics of this process that you should remember. unless some barrier. the sucrose will be uniformly distributed throughout the solution in the beaker (Fig. After a while. Since the sucrose concentration is initially high near the bottom of the beaker. at first more sucrose molecules will move toward the top of the vessel than toward the bottom. A concentration gradient is defined as the difference in the concentrations of a substance at two separate points divided by the distance between the points. 2-2B). At any given instant. When this occurs there are no longer any differences in concentration. Initially. However. • • When a difference in concentration exists diffusion will always occur. like an impermeable membrane. Figure 2-2.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. some sucrose will move toward the sides of the beaker. and d is the distance between the two points. and the system will have reached equilibrium. 2-2B and C). AND MEMBRANE PERMEABILITY preferentially in any direction. some toward the top. Eventually. Saturday. is placed in the way. This molecular movement is passive and spontaneous. In practice. If a lump of sucrose is placed in a beaker of water (Fig. and some toward the bottom. at first there will be unequal concentrations of sucrose in different parts of the vessel (Fig. 2-2D). OSMOSIS. The energy for the movement is contained in the concentration difference and does not need to be supplied from the outside. or (c1 – c2)/d. and the molecules will diffuse spontaneously from the region of high concentration to the region of lower concentration until they reach equilibrium. when concentrations are not the same we say there is a concentration gradient between the two regions. for diffusion in unstirred liquid systems tends to be surprisingly slow. more molecules will have moved toward the top of the vessel. No work need be done to cause diffusion to occur. and the concentration gradient will have diminished. 2-2A). where c1 and c2 are the concentrations at points 1 and 2. some of the sucrose will dissolve in the water adjacent to it. 2009 Page 20 7:05:05 PM . This will result in a gradient of sucrose in the solution in the beaker (Fig. Dissolution of sucrose molecules from a lump of sugar dropped into a beaker of water. January 24.
water concentration is high and vice versa. The higher the temperature of the system. However. When solute concentrations differ. both in terms of solute and solvent. This movement of water decreases the difference between concentrations on the two sides of the membrane. and temperature as follows: • • • Compounds with high molecular weights diffuse more slowly than compounds with low molecular weights. A semipermeable membrane acts as a selective barrier. the greater the rate of diffusion. For example. But there are always opposing forces… Saturday. the water would freely diffuse until the concentrated solution had expanded in volume and the dilute solution had shrunk in volume enough so that the concentration of solute (and water) on both sides of the membrane would be the same. where solute concentration is low. if water can move freely through the membrane. it moves into the compartment having the greatest concentration of solute (and lowest concentration of water). The osmotic pressure of a solution is the opposing hydrostatic force developed by the difference in water concentration between two solutions.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. the diffusion of water through a semipermeable membrane. concentration gradient. AND MEMBRANE PERMEABILITY The rate of diffusion is affected by molecular weight. 2009 Page 21 7:05:05 PM . even though their concentrations differ. permitting some substances (in this case. a membrane impermeable to solutes will prevent them from moving from a region of high concentration to a region of lower concentration. You will also observe evidence of osmosis. the greater the rate of diffusion. water) to pass through it but not permitting others (in this case. If there were no opposing forces. Osmosis is a type of diffusion that occurs when something prevents solute molecules from diffusing freely. ∆C × P × A MW × ∆X These and other ideas are summarized in Fick's law of diffusion: Q= where Q = net rate of diffusion ∆C = concentration gradient of substance P = permeability of the substance A = surface area of membrane MW = molecular weight of the substance ∆X = thickness of membrane In the first part of this exercise you will investigate the effects of molecular weight and concentration difference on the diffusion of solute molecules. Although the diffusion of water is denoted by the special name osmosis. OSMOSIS. certain solutes) to penetrate. The greater the concentration gradient. Water will diffuse from a region where its concentration is high (low solute concentration) to one where its concentration is low (high solute concentration). January 24. so do water concentrations. this process is simply a result of the tendency of molecules to move from a region of higher concentration to a region of lower concentration.
and there is no net flow of water through the membrane. the difference in hydrostatic pressure between compart- Saturday. 2009 Page 22 7:05:05 PM . January 24. the concentration of solute will now be higher in 1 (which has 6 g in 50 mL) than in 2 (which has 4 g in 50 mL). At equilibrium." Figure 2-3. and osmotic pressure is reported in units of hydrostatic pressure. OSMOSIS. As the volume in compartment 1 increases due to osmosis. The simplest way to do this is to this is to realize that the hydrostatic pressure difference is directly proportional to the difference in height between the two solutions at equilibrium and to report the pressure in units of "cm of water pressure. But here's where the opposing force comes in. the concentration of water will be higher in side 2 than in side 1.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. and water will move by osmosis from 2 to 1 (Fig. Here we imagine a beaker containing 100 mL of water divided into two compartments by a semipermeable membrane. At equilibrium. its level rises (Fig. 2-3. 2-3A). Effect of solute concentration on water distribution when a membrane is permeable to solvent but not to solute. This results in more hydrostatic pressure in compartment 1 than in compartment 2. the hydrostatic pressure in compartment 1 is equal and opposite to the osmotic pressure created by the difference in water concentration between the two compartments. If 6 g of a solute to which the membrane is not permeable are added to compartment 1 and 4 g of the same solute are added to compartment 2. Thus at equilibrium the hydrostatic pressure is a means of measuring the osmotic pressure. Consequently. 23B). in spite of the fact that the two compartments still have different solute concentrations. and this opposes the osmotic flow of water from compartment 2 to compartment 1 by physically pushing the water back from compartment 1 to compartment 2. AND MEMBRANE PERMEABILITY Let us look at an illustrative example in Fig.
the solution is hypertonic to the cell.9% NaCl solution. as a result of liver or kidney disease or severe malnutrition) the osmotic pressure of the blood decreases. Diffusion and osmosis are important processes in the body. to a solution that is 1 M in both glucose and sucrose. If the solute concentration in the external solution is lower than the concentration of solutes inside the cell. On the other hand. if a cell is in a hyperosmotic solution. Some (but not all) molecules are transferred from the environment into the blood (for instance. If a cell shrinks when it is placed in a solution. Thus a 2 M solution of glucose (Mr = 180) is osmotically equivalent to a 2 M solution of sucrose (Mr = 342). then the cell must be hyposmotic to the solution. the external solution is hyperosmotic to the cell.) Note that when comparing osmotic pressures. (This information will be useful to you when you formulate predictions for the experiments you will do in this exercise. If solute concentration is the same inside and outside of the cell. in the intestine) or from blood into the environment (as in the lungs) by diffusion. consequently. This transfer of molecules is very effective over extremely short distances (a fraction of a millimeter or less). the number of moles of particles of solute per liter. Solutions can be said to have osmolarity. the cell is in an isosmotic solution. the effect of the concentration of extracellular solutes on a cell's volume depends on whether or not the cell membrane is permeable to the solutes in question.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION.. centimeters) diffusion is too slow to deliver much material. AND MEMBRANE PERMEABILITY ments 1 and 2 is equal and opposite to the osmotic pressure brought about by the difference in solute and solvent concentrations. then the solution is hypotonic to the cell. OSMOSIS. For instance. diffusion causes the movement of some molecules from the blood into a cell or from a cell into the blood.g. over longer distances (e. the cell is in a hyposmotic solution. As you will see. without regard to chemical identity or charge. since the solute can quickly pass through the membrane and establish equal Saturday. that is. isosmotic. If a cell swells (and perhaps bursts) in a solution. mOsM. For example. a hyperosmotic solution of a solute that can penetrate the cell membrane might not make the cell shrivel. a 0. January 24. and also to a 1 M solution of NaCl (Mf = 58). the external fluid is said to be hyperosmotic to the cell. Osmolarity is measured in units of OsM. it is important to be clear about the point of reference. Note also that osmotic pressure is a function of the concentration of particles in solution. and the result is the movement of water out of the blood and accumulation of fluid in the tissues (edema). they are technically not interchangeable. In addition. If the concentration of solute molecules surrounding a cell is greater than the fluid inside the cell. Although these three terms are often used synonymously with hyperosmotic. etc. charge or molecular mass. Three additional terms describe the effects of solutions on cell volume. without regard to chemical identity.9% NaCl solution would be isosmotic to red blood cells. Osmosis is responsible for the absorption of much water from the intestine and in the kidney (which would otherwise produce urine at an embarrassing rate!). the surrounding solution is isotonic. 2009 Page 23 7:05:05 PM . The concentration of solutes in red blood cells (erythrocytes) is osmotically equivalent to that of a 0. and hyposmotic. and if there is no change in cell volume. However. if the concentration of blood proteins decreases (for instance.
however. January 24. the cell may actually swell with solvent under these conditions. This is why it is necessary to have two separate sets of terms. each containing 15 mL of agarose. Record both the distance and the time (T1) of your observation (Table 2-1). ruthenium red (786 g mol–1). Record the time (T0) in Table 2-1. Saturday. stabilized by the agarose to prevent mixing and convection. 5. The four compounds are methyl red (269 g mol–1). No image of letters is formed on your retina when you view newsprint through a suspension of intact red blood cells. Add 5 mL of one of the dye solutions to the top of each agarose column. In this exercise you will use solutions of four different compounds at the same concentration (1 mM).300 g mol–1). Mark the interface with a piece of tape or a marker. the bursting of cells that results is termed hemolysis. Observe that the dye molecules begin to move into the agarose as time passes. 2. You will take advantage of this fact to determine whether or not test solutions are hypotonic to red blood cells. Repeat the measurement 6 to 12 hr after the end of your laboratory period (T2) and then once each day for three more days (T3. fail to scatter light. OSMOSIS. Obtain four test tubes. At the end of the lab period measure the distance in mm between the top of the agarose and the farthest point at which you can see the color. In fact. one to compare osmotic pressures inside and outside the cell and one to describe a solution's effect on cells. a polysaccharide that forms a gel in aqueous media. This is essentially a column of water. since the cell has a high concentration of proteins within the cell that cannot pass through the membrane. A suspension of intact red blood cells will scatter light passing through it. This makes it possible to follow the progress of the molecules as they diffuse in a translucent medium. When red blood cells are placed in a hypotonic solution. therefore. PROCEDURE: EFFECT OF MOLECULAR MASS ON RATE OF DIFFUSION 1.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. methylene blue (374 g mol–1). since the released hemoglobin forms a clear solution. 2009 Page 24 7:05:05 PM . Note that the interface between the colored solution and the agarose is sharp. T4. All of these compounds are brightly colored. 4. AND MEMBRANE PERMEABILITY concentrations on both sides. erythrocytes placed in a hypotonic solution will swell and burst and will. and alcian blue (1. no mixing occurs. 3. but a clear image appears when newsprint is examined through a suspension in which hemolysis has taken place. T5). Record your observations in Table 2-1.
OSMOSIS. Draw tangents to your curves at T0. and calculate their slopes: these are the initial rates of diffusion at T0 in mm day–1. or symbols represent. Include a legend that explains what the different colors. Your graph should include four lines. lines. it is here that you would expect to get your maximum rate of diffusion. 2009 Page 25 7:05:05 PM . so graph it on the abscissa (X-axis). Time is your independent variable. AND MEMBRANE PERMEABILITY TABLE 2-1. Prepare a graph showing the effect of molecular mass on diffusion rate. either hand-drawn (boring!) or using spreadsheet software such as Excel® or SigmaPlot®. such as concentration and temperature. or different symbols. Make a new graph of initial rates of diffusion as a function of the molecular masses of the four dyes. your dependent variable. What is the relationship? Is Fick's law obeyed? How long would it take each of your dyes to diffuse 1 cm? Saturday. using mm as your unit of measurement. EFFECT OF MOLECULAR MASS ON RATE OF DIFFUSION Methyl Red (269 g mol–1) Observation Number Date and Time Hours Elapsed Migration of Dye (mm) Methylene Blue (374 g mol–1) Date and Time Hours Elapsed Migration of Dye (mm) Ruthenium Red (786 g mol–1) Date and Time Hours Elapsed Migration of Dye (mm) Alcian Blue (1. and graph migration distance. have been successfully held equal. different kinds of lines. using hours as your unit of measurement. either by using different colors. or does it slow down? The concentration gradient is sharpest at T0. January 24. Is the rate of diffusion (the slope of your lines) constant during your observation period.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. or some combination thereof. your results in Table 2-1 would be expected to show how molecular size affects diffusion.300 g mol–1) Date and Time Hours Elapsed Migration of Dye (mm) 0 1 2 3 4 5 ANALYSIS: EFFECT OF MOLECULAR MASS ON RATE OF DIFFUSION If the other variables. on the ordinate (Y-axis). Distinguish the four lines. one for each of the dyes.
2009 Page 26 7:05:05 PM . There will be no cells to scatter the light and the solution will become transparent. This is your stock erythrocyte suspension and it is turbid because the cells scatter light. What is the relationship? Is Fick's law obeyed? PROCEDURE: OSMOSIS 1. with time the cells will settle to the bottom of the tube. The first will of course only have three lines.3 mM) Observation Number Date and Time Hours Elapsed Migration of Dye (mm) Methylene Blue (1 mM) Date and Time Hours Elapsed Migration of Dye (mm) Methylene Blue (3 mM) Date and Time Hours Elapsed Migration of Dye (mm) 0 1 2 3 4 5 ANALYSIS: EFFECT OF CONCENTRATION GRADIENT ON RATE OF DIFFUSION Make a pair of graphs similar to the ones you made in the previous part. so you need to keep the stock erythrocyte suspension gently mixed. OSMOSIS.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. and plot their values (initial rates) as a function of concentration of dye in mM. since there are only three concentrations of methylene blue.9% NaCl). January 24. If the cells gain water they will swell and rupture. However. 2. TABLE 2-2: EFFECT OF CONCENTRATION GRADIENT ON RATE OF DIFFUSION Methylene Blue (0. and record your observations in Table 2-2. Add the different methylene blue solutions to the top of the agarose column. 1 mM. AND MEMBRANE PERMEABILITY PROCEDURE: EFFECT OF CONCENTRATION GRADIENT ON RATE OF DIFFUSION You will compare the diffusion rates of three solutions of methylene blue at different concentrations (0. Take slopes at T0 again. Obtain three test tubes containing agarose. Make the same measurements as you made in the previous section. Saturday. and 3 mM). If you place a piece of paper with typed words behind the erythrocyte suspension you cannot distinguish any letters. Obtain a whole blood sample and dilute 20 drops of blood into 10 mL of isotonic saline (0.3 mM. 3. 1.
For uncharged compounds. Membrane permeability. Add 3 drops of stock erythrocyte suspension and mix. January 24. 5. In Table 2-3 describe the turbidity of the three solutions. hypertonic or hypotonic. In general.9% Saline 10% Saline Appearance of suspension Microscopic appearance of cells Tonicity of solution PART II: MEMBRANE PERMEABILITY INTRODUCTION The passive movement of materials across cell membranes is the result of a) the permeability of the membrane to the diffusing substance and b) the driving force. 4. The penetration rate of a cell membrane by a molecular species via diffusion is important in determining the chemical composition of the cell. Get three 10 x 100-mm test tubes and add 2 mL of deionized water. TABLE 2-3: OSMOSIS (RESULTS) Three drops stock erythrocyte suspension in 2 mL of Deionized water 0. Place a few drops of each solution on a microscope slide and note the appearance of the cells. You might want to check the appearance of your stock solution for comparison. In this exercise. AND MEMBRANE PERMEABILITY 2. This representation is referred to as the fluid mosaic model. in many cases. or 10% saline to each. 3. In the case of ionic diffusion. Saturday. A substance to which the membrane is impermeable will not be found in the cytoplasm unless it is produced there or actively transported into the cytoplasm. active transport. 2-4. the driving force is a concentration gradient between the inside and the outside of the cell. The structure of a cell membrane is shown in Fig. the driving force results from the concentration gradient and the electrical potential difference between the cell's interior and its exterior. these constituents are free to move within the membrane. and.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. we will investigate the effects of lipid solubility and molecular weight on the permeability of red blood cell membranes. and they are completely impermeable to many compounds. OSMOSIS. 0. biological membranes are far less permeable than an equivalent thickness of solvent. because the membrane is a mosaic of phospholipids and proteins. Label the tubes. 2009 Page 27 7:05:05 PM .9% saline. Identify which solutions were isotonic. and driving force determine the rate of movement of nutrients into and waste compounds out of the cell.
January 24.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. 2009 Page 28 7:05:05 PM . phosphate group attached to a glycerol backbone (Fig. Intrinsic proteins partially or fully penetrate the membrane and may serve as transport or receptor molecules. Fluid mosaic model of a cell membrane composed of a lipid bilayer with proteins "floating" in the membrane. 2-5). When they are surrounded by water molecules. charged. called a lipid bilayer. this results in two layers of phospholipids. sugars. The hydrocarbon interior of the membrane constitutes a barrier to penetration of polar solutes. while the hydrophobic (water-avoiding) hydrocarbon chains point away from the water. The nonpolar hydrophobic portion of each phospholipid molecule is directed toward the center of the membrane. and ions. The cell membrane is a thin membrane of phospholipids studded with proteins. In the membranes of cells and organelles. Phospholipids consist of two fatty acid chains and one polar. Saturday. such as amino acids. the hydrophilic (meaning "water-loving") ends of the phospholipid molecules face outward toward the water. and the polar hydrophilic portion is directed toward the water environment either outside or inside the cell. AND MEMBRANE PERMEABILITY Figure 2-4. OSMOSIS.
the rate of diffusion across cell membranes decreases as molecular size increases (that is. Therefore. The inside of the tube is lined with hydrophilic groups. These polar solutes must take specific avenues through lipid membranes. Molecules of low lipid solubility can pass through pores in the lipid membrane if they are small. those with high lipid solubility will enter the cell more readily than those with low lipid solubility. The simplest way for a polar solute to cross a cell membrane is by way of pores. A more complicated mechanism by which polar solutes enter cells involves membrane carriers. Most of the substances a cell comes in contact with have charged groups. although the permeability of membranes is highly variable among organisms. Some solutes that are not lipid soluble can penetrate the cell membrane with the aid of proteins in the membrane. small molecules enter the cell faster than large molecules). polar solutes can diffuse through the membrane in aqueous solution. all covalently joined.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. composed of two fatty acid chains. January 24. facing the phospholipids are many hydrophobic groups. are usually oriented so that the polar portion extends towards one end of the molecule and the two fatty acid chains towards the opposite. Thus. 2009 . glycerol. Therefore. OSMOSIS. within a group of chemically related. On the outside of the tube. two generalizations seem to be valid for all cells: • For chemically related water-soluble compounds. or channels. nonpolar compounds. The mechanism by which a solute in the extracellular fluid enters a cell depends on the polarity of the solute. they cannot pass through the lipid portions of membranes. Phospholipids. allowing the channel to be filled with water. large molecules of low lipid solubility will be excluded. On the other hand. Nonpolar solutes pass directly through the membrane's lipid matrix. so they are often represented diagrammatically as a polar ball with a nonpolar tail. phosphoric acid. and they are water-soluble. AND MEMBRANE PERMEABILITY Figure 2-5. and an alcohol such as choline. To summarize. A channel is a tube through a membrane formed by one or more intrinsic membrane proteins. Page 29 7:05:05 PM Saturday. which undergo a cycle of binding and conformational change as solutes move across the membrane.
the effect of solute molecules in the solution surrounding a cell will depend on whether or not the membrane is permeable to them. When hemolysis occurs. Consequently.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. This influx of water causes the cell to swell. The faster a solute diffuses into the cell. When solute enters. hemolysis can also take place when a solute in the extracellular solution moves down its concentration gradient and enters a cell. If the membrane is not permeable to solute. escape. it may not be seen at all if the suspension is dense. the solutions have the same osmotic pressure as the cell interior). if the solute molecules in the external solution are not found inside the cell. Thus the solution is isotonic with respect to the cell. when the cells hemolyze. the time taken for the solution to clear gives us a measure of membrane permeability to the solute in question. OSMOSIS. the rate of diffusion across cell membranes increases with increasing lipid solubility and is (more or less) independent of molecular size. nonpolar compounds. In this case the solution is hypotonic with regard to the cell. For the reasons given in the preceding paragraph. increasing the internal osmotic pressure. Because cell membranes are selectively permeable. we need to distinguish between the osmotic pressure of a solution and its effect on cells. Recall that a suspension of blood cells (in plasma or in physiological saline) scatters light. but each of the compounds have Saturday. However. including hemoglobin. Imagine that a cell is placed a solution having a total solute concentration equivalent to the concentration of solutes within the cell. One of the best known preparations for studying membrane permeability is the mammalian erythrocyte. First you will determine what molarity of NaCl is isosmotic to erythrocytes. a thread or a piece of newsprint viewed through a suspension of blood cells will appear faint and fuzzy. because of the higher concentration of solute outside the cell. into the cell. AND MEMBRANE PERMEABILITY • For chemically related. it will enter the cell. A sharp image of a small object viewed through the suspension cannot be formed. Then you will use compounds that have been prepared in isosmotic solutions (that is. To measure hemolysis time it is simply necessary to measure in seconds the time it takes for the cells to rupture and for the suspension to become transparent. We will record hemolysis time and calculate the hemolysis rate. the solution becomes translucent and a sharp image can be seen. January 24. providing that the driving force remains the same. We will take advantage of this process to compare the permeability of the erythrocyte membrane to different solutes and water. and it may burst. 2009 Page 30 7:05:05 PM . The cell is therefore in an isosmotic solution. if the solute can cross the membrane. Hemolysis rate is defined to be the reciprocal of the hemolysis time. the sooner it will hemolyze. even though the solution is isosmotic. the osmotic pressure inside the cell increases. so divide the hemolysis time into 1 to obtain the rate in units of sec–1. or are found in lower concentrations. Thus. there will be no net movement of water into or out of the cell. However. This is the basis of our method for following the diffusion of solutes from the external bathing medium. and water enters the cell. the internal solutes. In Part I you saw that hemolysis occurs when red blood cells are placed in deionized water. However.
2009 Page 31 7:05:05 PM .07 M. 5. Before beginning the experiments that follow. and check the solution for clarity. add three drops of your well-mixed stock blood suspension to the first tube. The cells are shaped as biconcave discs (doughnuts of sorts with a thin section where the hole would be) (a bialy. and 0. they are unusual in some regards. Before you begin these experiments it is worthwhile to practice measuring hemolysis time using the stock erythrocyte solution from the previous experiment. January 24. Repeat the entire procedure until you are comfortable with your ability to accurately measure hemolysis time. Repeat with each of the other tubes.05 M. OSMOSIS. 0. for those of you who know from bialys). Record the time (in seconds) at which the solution clears. and 1/60 sec–1 a maximal estimate of the hemolysis rate for such a sample. if you have one.06 M. 4.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. 2. mix rapidly. PROCEDURE: PERMEABILITY TO WATER 1. The solution will become transparent in a few seconds (record the hemolysis time in Table 2-4 and calculate the rate). Do no wait for more than a minute. The time elapsed is the hemolysis time.10 M NaCl solutions will be available. A hemolysis time of 60 seconds thus becomes a minimal estimate of the hemolysis time. Add 3 drops of stock erythrocyte solution.09 M. 0. This shape gives them flexibility—the portion of the membrane that usually dips in the center can also flex out. be sure you understand why hemolysis time can be used as an indicator of membrane permeability and how solutes in an isosmotic solution can cause hemolysis to occur. note the time and mix. Keep this in mind during your experimental work. it probably won't occur at all. Solutions that are somewhat hyposmotic but not too hyposmotic can cause water to enter the cell and cause it to balloon out but not break. 3. Add 2 mL of deionized water to a test tube. Expect to see changes within seconds. To each tube in the hemolysis apparatus add 2 mL of one of the NaCl solutions. Repeat two times and report the average of the three hemolysis times. You will investigate how these two characteristics alter hemolysis time and rate. 0. This procedure is best done with a stopwatch. Noting the exact time of addition.08 M. Your hemolysis apparatus will consist of a test tube rack placed in good light and either a string or a piece of paper with writing that will come into sharp focus as lysis occurs. You also should be aware that although erythrocytes are the cells of choice for this type of demonstration. AND MEMBRANE PERMEABILITY different molecular weights and lipid solubilities. if hemolysis has not occurred within that time. Therefore these cells can stretch a certain amount. and calculate the rate. 0. 0. Saturday.
9% NaCl.e. January 24..00 M 0. 2009 . Saturday. You will now repeat the previous protocol with several alcohol solutions that are 0. Obtain a whole blood sample and dilute 20 drops to 10 mL 0.05 M 0.02 M in NaCl. i.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION.10 M 0. Because the cells tend to settle to the bottom of the tube it is necessary to mix the stock before removing any material.15 M 0. OSMOSIS.20 M ANALYSIS: PERMEABILITY TO WATER At what concentration do the NaCl solutions appear to become isotonic.3 M in alcohol and 0. at what concentration does the hemolysis time first become >120 seconds? What is the molarity of the famous 0.9% NaCl physiological saline solution? Does this agree with your results? PROCEDURE: PERMEABILITY TO VARIOUS ALCOHOLS 1. Thus the solutions are Page 32 7:05:05 PM 2. This is your stock erythrocyte solution. As you know. These alcohol solutions have the same initial solute and water concentration as mammalian cytoplasm but the alcohols are not normally present in the cytoplasm of normal cells. it is turbid because the cells scatter light. AND MEMBRANE PERMEABILITY TABLE 2-4: PERMEABILITY TO WATER (RESULTS) Concentration of NaCl solution Hemolysis time (sec) Average hemolysis time (sec) Hemolysis rate (sec–1) 0.
The time elapsed is the hemolysis time. For each of the alcohols (methanol. ethanol. Record the time (in seconds) at which the solution clears. Add 2 mL of each alcohol solution to separate 10 x 100-mm test tubes and label the tubes. Repeat two times and report the average of the three hemolysis times in Table 2-5 and calculate the rates of hemolysis. January 24. 2009 Page 33 7:05:05 PM . the rate of diffusion of these alcohols across the erythrocyte membrane should be directly proportional to the concentration gradient of the alcohol (constant in this experiment). OSMOSIS. and check the solution for clarity. mix rapidly. propanol and butanol) perform the following in triplicate. Noting the exact time of addition.000 6. AND MEMBRANE PERMEABILITY isosmotic with cells.000 OH OH OH According to Fick's law. add three drops of your well-mixed stock blood suspension to the first tube.BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. TABLE 2-5: PERMEABILITY TO VARIOUS ALCOHOLS (RESULTS) Alcohol Hemolysis time (sec) Average hemolysis time (sec) Hemolysis rate (sec–1) Methanol Ethanol Propanol Butanol ANALYSIS: PERMEABILITY TO VARIOUS ALCOHOLS TABLE 2-6: PROPERTIES OF THE FOUR ALCOHOLS Alcohol Methanol Ethanol n-Propanol n-Butanol Molecular mass 32 46 60 74 Structure OH Lipid solubility 80 350 1. the permeability of the alcohol (a variable in this experiSaturday.
OSMOSIS. Make the plot. The rate is inversely proportional to the molecular mass of the substance (a variable in this experiment) and the thickness of the membrane (a constant in this experiment).BIOLOGY 204: INTRODUCTION TO PHYSIOLOGY — LABORATORY MANUAL EXERCISE 2: DIFFUSION. If you see something different. the permeabilities are exerting an effect. 2009 Page 34 7:05:05 PM . you should see a steadily decreasing curve. If the permeabilities of the four alcohols are all the same. something like what you saw in Part I. AND MEMBRANE PERMEABILITY ment). To sort out the effects of two variables. one can plot the rate of diffusion (rate of hemolysis) against the molecular masses of the four alcohols. January 24. Do your results support the hypothesis that the permeabilities of these alcohols are function of their lipid solubilities? Saturday. and the surface area of membrane (constant in this experiment).
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